Methods for damaging cells using effector functions of anti-gfra1 antibodies

ABSTRACT

The present invention relates to the use of cytoxicity based on the effector function of anti-GFRA1 antibodies. Specifically, the present invention provides methods and pharmaceutical compositions that comprise an anti-GFRA1 antibody as an active ingredient for damaging GFRA1-expressing cells using antibody effector function. Since GFRA1 is strongly expressed in breast, gastric, liver, renal or lung cancer cells, the present invention is useful in breast, gastric, liver, renal or lung cancer therapies.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. National Phase of PCT/JP2005/004859, filedMar. 11, 2005. The contents of the aforementioned application is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to methods for damaging cells using theeffector function of anti-GFRA1 antibodies, or to compositions for thispurpose.

BACKGROUND ART

Breast cancer, a genetically heterogeneous disease, is the most commonmalignancy in women. An estimation of approximately 800000 new cases wasreported each year worldwide (Parkin D M, Pisani P, Ferlay J (1999). CACancer J Clin 49: 33-64). Mastectomy is the first concurrent option forthe treatment of this disease. Despite surgical removal of the primarytumors, relapse at local or distant sites may occur due to undetectablemicrometastasis (Saphner T, Tommey D C, Gray R (1996). J Clin Oncol, 14,2738-2749.) at the time of diagnosis. Cytotoxic agents are usuallyadministered as adjuvant therapy after surgery aiming to kill thoseresidual or premalignant cells.

Treatment with conventional chemotherapeutic agents is often empiricaland is mostly based on histological tumor parameters, and in the absenceof specific mechanistic understanding. Target-directed drugs aretherefore becoming the bedrock treatment for breast cancer. Tamoxifenand aromatase inhibitors, two representatives of its kind, have beenproved to have great responses used as adjuvant or chemoprevention inpatients with metastasized breast cancer (Fisher B, Costantino J P,Wickerham D L, Redmond C K, Kavanah M, Cronin W M, Vogel V, Robidoux A,Dimitrov N, Atkins J, Daly M, Wieand S, Tan-Chiu E, Ford L, Wolmark N(1998). J Natl Cancer Inst, 90, 1371-1388; Cuzick J (2002). Lancet 360,817-824). However the drawback is that only patients expressed estrogenreceptors are sensitive to these drugs. A recent concerns were evenraised regarding their side effects particularly lay on the possibilityof causing endometrial cancer for long term tamoxifen treatment as wellas deleterious effect of bone fracture in the postmenopausal women inaromatase prescribed patients (Coleman RE (2004). Oncology. 18 (5 Suppl3), 16-20). Owing to the emergence of side effect and drug resistance,it is obviously necessarily to search novel molecular targets forselective smart drugs on the basis of characterized mechanisms ofaction.

Gastric cancer is a leading cause of cancer death in the world,particularly in the Far East, with approximately 700,000 new casesdiagnosed worldwide annually. Surgery is the mainstay in terms oftreatment, because chemotherapy remains unsatisfactory. Gastric cancersat an early stage can be cured by surgical resection, but prognosis ofadvanced gastric cancers remains very poor.

Hepatocellular carcinoma (HCC) is one of the most common cancersworldwide and its incidence is gradually increasing in Japan as well asin United States (Akriviadis E A, et al., Br J. Surg. 1998 October;85(10):1319-31). Although recent medical advances have made greatprogress in diagnosis, a large number of patients with HCCs are stilldiagnosed at advanced stages and their complete cures from the diseaseremain difficult. In addition, since patients with hepatic cirrhosis orchronic hepatitis have a high risk to HCCs, they may develop multipleliver tumors, or new tumors even after complete removal of initialtumors. Therefore development of highly effective chemotherapeutic drugsand preventive strategies are matters of pressing concern.

Renal cell carcinoma (RCC) is the third most common malignancy of thegenitourinary system and corresponds to 2-3% of all human malignancies.Surgical resection is the most effective treatment for patients withlocalized RCC tumors, but such treatment for patients withadvanced-stage RCC is not satisfactory. Although some biomedicaltherapies have been reported to show 20% response rate, they often causesevere adverse reactions and do not generally improve patients'survival. Among patients who have surgical treatment, approximately25-30% relapse after surgery (Ljungberg B., Alamdari F. I., Rasmuson T.& Roos G. Follow-up guidelines for nonmetastatic renal cell carcinomabased on the occurrence of metastases after radical nephrectomy. BJUInt. 84, 405-411 (1999); Levy D A., Slaton J W., Swanson D A. & Dinney CP. Stage specific guidelines for surveillance after radical nephrectomyfor local renal cell carcinoma. J. Urol. 159, 1163-1167 (1998)). Tumorstage and surgical respectability are the most important prognosticfactors for RCC; however, to date, little is known of the underlyingmolecular mechanisms that influence this variety in prognoses.

RCC tumors can be subdivided on the basis of histological features intoclear cell (80%), papillary (˜10%), chromophobe (<5%), granular, spindleand cyst-associated carcinomas (5-15%). Each of these histologicalsubtypes shows unique clinical behavior, with clear-cell and granulartypes tending to show more aggressive clinical phenotypes.

Lung cancer is one of the most common lethal human tumors.Non-small-cell lung cancer (NSCLC) is the most common form, accountingfor nearly 80% of lung tumors (American Cancer Society, Cancer Facts andFigures 2001, Am. Chem. Soc. Atlanta, 2001). The majority of NSCLCs arenot diagnosed until an advanced stage, and thus the overall 10-yearsurvival rate has stayed low at 10%, despite recent advances inmultimodality therapies (Fry et al., Cancer, 86: 1867-76, 1999).Currently, chemotherapy using platinum is considered to be a fundamentaltherapy for NSCLCs. However, the therapeutic action of pharmaceuticalagents has not progressed beyond the point of being able to prolong thesurvival of advanced NSCLC patients to a certain extent (Chemotherapy innon-small cell lung cancer: a meta-analysis using updated data onindividual patients from 52 randomized clinical trials, Non-small CellLung Cancer Collaborative Group, Bmj. 311: 899-909, 1995). A number oftargeting therapies are being investigated, including those that usetyrosine kinase inhibitors. However, to date, promising results havebeen achieved only in a limited number of patients, and in somepatients, therapeutic effects have accompanied severe side effects (Kriset al., Proc Am Soc Clin Oncol, 21: 292a (A1166), 2002).

Research aiming at the elucidation of carcinogenic mechanisms hasrevealed a number of candidate target molecules for anti-tumor agents.For example, the farnesyltransferase inhibitor (FTI) is effective in thetherapy of Ras-dependent tumors in animal models (Sun J, et al.,Oncogene. 1998 March; 16(11):1467-73). This pharmaceutical agent wasdeveloped to inhibit growth signal pathways related to Ras, which isdependant on post-transcriptional farnesylation. Human clinical trialswhere anti-tumor agents were applied in combination with the anti-HER2monoclonal antibody trastuzumab with the aim of antagonizing theproto-oncogene HER2/neu have succeeded in improving clinical response,and improved the overall survival rate of breast cancer patients (MolinaM A, et al., Cancer Res. 2001 Jun. 15; 61(12):4744-9). Tyrosine kinaseinhibitor STI-571 is an inhibitor which selectively deactivates bcr-ablfusion protein. This pharmaceutical agent was developed for the therapyof chronic myeloid leukemia, where the constant activation of bcr-abltyrosine kinase has a significant role in the transformation of whiteblood cells. Such pharmaceutical agents are designed to inhibit thecarcinogenic activity of specific gene products (O'Dwyer M E & Druker BJ. Curr Opin Oncol. 2000 November; 12(6):594-7). Thus, in cancer cells,gene products with promoted expression are usually potential targets forthe development of novel anti-tumor agents.

Another strategy for cancer therapy is the use of antibodies which bindto cancer cells. The following are representative mechanisms ofantibody-mediated cancer therapy:

Missile therapy: in this approach a pharmaceutical agent is bound to anantibody that binds specifically to cancer cells, and the agent thenacts specifically on the cancer cells. Even agents with strong sideeffects can be made to act intensively on the cancer cells. In additionto pharmaceutical agents, there are also reports of approaches whereprecursors of pharmaceutical agents, enzymes which metabolize theprecursors to an active form, and so on are bound to the antibodies.

The use of antibodies which target functional molecules: this approachinhibits the binding between growth factors and cancer cells using, forexample, antibodies that bind growth factor receptors or growth factors.Some cancer cells proliferate depending on growth factors. For example,cancers dependent on epithelial growth factor (EGF) or vascularendothelial growth factor (VEGF) are known. For such cancers, inhibitingthe binding between a growth factor and cancer cells can be expected tohave a therapeutic effect.

Antibody cytotoxicity: antibodies that bind to some kinds of antigenscan comprise cytotoxicity to cancer cells. With these types ofantibodies, the antibody molecule itself comprises a direct anti-tumoreffect. Antibodies that display cytotoxicity to cancer cells are gainingattention as antibody agents expected to be highly effective againsttumors.

DISCLOSURE OF THE INVENTION

The present inventors investigated antibodies able to induce cytotoxity,targeting genes showing increased expression in cells. The resultsrevealed that potent cytotoxicity can be induced in GFRAL1-expressingcells when those cells are contacted with anti-GFRA1 antibodies, thuscompleting the present invention.

Specifically, the present invention relates to the followingpharmaceutical compositions or methods:

[1] Pharmaceutical compositions comprising an anti-GFRA1 antibody as anactive ingredient, wherein the anti-GFRA1 antibody damages (i.e., killsthe cell, is toxic to the cell, or otherwise inhibits growth or celldivision), an GFRA1-expressing cell using the antibody effectorfunction.

[2] The pharmaceutical compositions are used to treat any pathologicalcondition associated with GFRA1-expressing cells. In typicalembodiments, the cell is a cancer cell, such as breast, gastric, liver,renal or lung cancer cell.

[3] The antibodies in the pharmaceutical compositions of the inventionare typically monoclonal antibodies.

[4] In some embodiments, the antibody of the invention comprises aneffector function such as antibody-dependent cytotoxicity,complement-dependent cytotoxicity, or both.

[5] Methods for damaging an GFRA1-expressing cell, will comprise thesteps of:

-   -   a) contacting the GFRA1-expressing cell with an anti-GFRA1        antibody. As a result of the binding of the antibody the        effector function of the antibody will cause damage (i.e.,        cytotoxicity) to the GFRA1-expressing cell.

[6] Immunogenic compositions for inducing an antibody that comprises aneffector function against an GFRA1-expressing cell. The compositionstypically comprise as an active ingredient, a GFRA1 polypeptide, animmunologically active fragment thereof, or a nucleic acid molecule theexpresses the polypeptides or fragments.

[7] Methods for inducing an antibody that comprises an effector functionagainst an GFRA1-expressing cell, wherein the method comprisesadministering a GFRA1 polypeptide, an immunologically active fragmentthereof, or a cell or a DNA that can express the polypeptides orfragments.

The present invention relates to pharmaceutical compositions fordamaging GFRA1-expressing cells using antibody effector function,wherein the compositions comprise as an active ingredient an anti-GFRA1antibody. The present invention also relates to uses of an anti-GFRA1antibody to produce pharmaceutical compositions for damagingGFRA1-expressing cells using the anti-GFRA1 antibody effector function.The pharmaceutical compositions of the present invention compriseanti-GFRA1 antibodies and pharmaceutically acceptable carriers. Thepresent inventors used cDNA microarrays for gene expression analysis ofbreast cancer cells and normal cells collected from breast cancerpatients.

A number of genes with specifically enhanced expression in breast cancercells were subsequently identified. Of these genes with alteredexpression in breast cancer cells, one gene, glial cell line-derivedneurotrophic factor (GDNF) family receptor alpha 1 (GFRA1) gene encodingcytoplasmic membrane protein with low levels of expression in majororgans was selected as a candidate target gene for cancer therapies. Byselecting genes with low levels of expression in major organs, it wasthought that the danger of side effects could be avoided. Among theprotein encoded by the genes selected in this way, anti-GFRA1 antibodieswere confirmed to have effector functions against GFRA1-expressingcells. In addition, a similar effect was confirmed in other cancer celllines, such as the gastric, liver, renal, and lung cancer cell linesthat this gene over-expressed.

The findings obtained by the present inventors show that, in a forcedexpression system, GFRA1 tagged with c-myc-His was localized incytoplasmic membrane, which was confirmed using Immuno-fluorescencemicroscopy. The GFRA1 gene encodes an amino acid sequence expected tocomprise a signal peptide at its N-terminal. As mentioned above, thisprotein was observed to be chiefly localized in the cytoplasmicmembrane, and thus it was thought to be a transmembrane protein. Inaddition, the low expression level of this gene in major organs, and itshigh expression in breast cancer cells, establishes that GFRA1 is usefulas a clinical marker and therapeutic target.

Conditions required for destroying cancer cells using effector functionare, for example, the following:

-   -   Expression of large numbers of antigenic molecules on the        membrane surface of cancer cells,    -   Uniform distribution of antigens within cancerous tissues,    -   Lingering of antigens bound to antibodies on the cell surface        for a long time.

More specifically, for example, antigens recognized by antibodies mustbe expressed on the surface of the cell membrane. In addition, it ispreferable that the ratio of antigen-positive cells is as high aspossible in cells forming cancerous tissues. In an ideal situation, allcancer cells are antigen-positive. When antigen-positive and negativecells are mixed in cancer cell populations, the clinical therapeuticeffect of the antibodies may not be expected.

Usually, when as many molecules as possible are expressed on the cellsurface, potent effector functions can be expected. It is also importantthat antibodies bound to antigens are not taken up into cells. Somereceptors are taken up into cells (endocytosis) after binding to aligand. Equally, antibodies bound to cell surface antigens can also betaken up into the cell. This kind of phenomenon, whereby antibodies aretaken up into cells, is called internalization. When internalizationoccurs, the antibody constant (Fc) region is taken up into the cell.However, cells or molecules essential to effector function are outsidethe antigen-expressing cells. Thus, internalization inhibits antibodyeffector function. Therefore, when expecting antibody effector function,it is important to select an antigen that causes less antibodyinternalization. The present inventors revealed for the first time thatGFRA1 is a target antigen possessing such a property.

“Effector function” in the present invention refers to cytotoxicityinvolved with the Fc regions of antibodies. Alternatively, functionsthat drive the effect whereby the Fc regions of antibodies bound toantigens damage cells comprising those antigens, can also be referred toas antibody effector function. Specifically, Antibody DependentCell-mediated Cytotoxicity (ADCC), Complement Dependent Cytotoxicity(CDC), and neutralizing activity are known as antibody effectorfunctions. Each function is described below.

Antibody Dependent Cell-Mediated Cytotoxicity (ADCC):

Cells exist which comprise Fc receptors specific to the Fc region ofimmunoglobulin classes IgG, IgE, or IgA. Cells that comprise acorresponding Fc receptor recognize and bind to antibodies bound to cellmembranes or so on. For example, an IgG class antibody is recognized byFc receptors on T cells, NK cells, neutrophils, and macrophages. Thesecells bind to and are activated by the Fc region of IgG classantibodies, and express cytotoxicity against cells to which theseantibodies have bound. Cells which acquire cytotoxicity via antibodyeffector function are called effector cells. ADCC may be divided basedon the type of effector cell, as follows:

ADMC: IgG-dependent macrophage-mediated cytotoxicity, and

ADCC: IgG-dependent NK-cell-mediated cytotoxicity.

There is no limitation on types of effector cells in the ADCC of thepresent invention. In other words, the ADCC of the present inventionalso comprises ADMC, where macrophages are the effector cells.

Antibody ADCC is known to be an important mechanism of the anti-tumoreffects, particularly in cancer therapies that use antibodies (NatureMed., 6: 443-446, 2000). For example, a close relationship between thetherapeutic effect of anti-CD20 antibody chimeric antibodies and ADCChas been reported (Blood, 99: 754-758, 2002). Thus ADCC is alsoparticularly important among antibody effector functions in the presentinvention.

For example, ADCC is thought to be an important mechanism in theanti-tumor effects of Rituxan, Herceptin, and so on, for which clinicalapplication has already begun. Rituxan and Herceptin are therapeuticagents for non-Hodgkin's lymphoma and metastatic breast cancer,respectively.

At present, the mechanism for ADCC-mediated cytotoxicity is roughlyexplained as follows: effector cells, which are bridged to target cellsvia antibodies bound to the cell surface, are thought to induce targetcell apoptosis by transmitting some sort of lethal signal to the targetcells. In any case, antibodies that induce cytotoxicity by effectorcells are comprised in the antibodies that comprise effector function ofthe present invention.

Complement Dependent Cytotoxicity (CDC):

The Fc regions of immunoglobulins bound to antigens are known toactivate complementary pathways. It has also been revealed that theactivation pathway may differ depending on the class of immunoglobulin.For example, of the human antibodies, IgM and IgG activate the classicalpathway. On the other hand, IgA, IgD, and IgE do not activate thispathway. The activated complements produce, via a number of reactions, aC5b-9 membrane attack complex (MAC) comprising cell membrane-damagingactivity. MACs generated in this way are thought to damage viralparticles and cell membranes, independently of effector cells. Themechanism for MAC-mediated cytotoxicity is based on the following. MACscomprise a strong binding affinity for cell membranes. MACs bound to acell membrane open a hole in the cell membrane, making it easy for waterto flow in and out of the cell. As a result, the cell membrane isdestabilized, or the osmotic pressure is changed, and the cell isdestroyed. Cytotoxicity due to an activated complement only extends tomembrane close to the antibody which has bound the antigen. For thisreason, MAC-mediated cytotoxicity is dependent on antibody specificity.ADCC and CDC can express cytotoxicity independent of each other.However, in practice, these cytotoxicities may function in composite inliving bodies.

Neutralizing Activity:

Antibodies exist which have the function of depriving infectivity ofpathogens and activity of toxins. Antibody-mediated neutralization canbe achieved by binding of an antigenic variable region to an antigen, orcan require complement mediation. For example, in some cases, anti-viralantibodies require complement mediation in order to deprive a virus ofits infectivity. Fc regions are essential to the participation ofcomplements. Thus, such antibodies comprise effector function thatrequires Fc for neutralizing viruses and cells.

In the present invention, effector function can also be explained as arole that determines the biological activity triggered by antigenrecognition of an antibody. Herein, preferable target cells are cancercells. In addition, effector cell functions carried out by the Fcregions of various antibodies rely heavily on antibody class. The Fcregion of IgG, IgE, and IgA class antibodies each binds to a specific Fcreceptor, and, for example, activates cells that have Fc receptors, andfunctions in intercellular antibody transport. In particular, IgG classantibodies activate effector cells via Fc receptors on these cells, andthen kill target cells to which the variable regions of the antibodiesare bound. This is called antibody-dependent cell-mediated cytotoxicity(ADCC). In ADCC, T cells, NK cells, neutrophils, macrophages, or suchfunction as effector cells. On the other hand, the function ofactivating complement is limited to IgM and IgG class antibodies.Particularly, the function of lysing cells to which antibody variableregions are bound is called complement-dependent cytotoxicity (CDC).

Of these, preferable effector functions herein are either ADCC or CDC,or both. The present invention is based on the finding that anti-GFRA1antibodies bind to GFRA1-expressing cells, and then express effectorfunction.

The present invention also relates to methods for damagingGFRA1-expressing cells, which comprise the following steps:

1) contacting the GFRA1-expressing cells with anti-GFRA1 antibodies, and

2) using the effector function of the antibodies which have bound to theGFRA1-expressing cells to damage the cells.

In the methods or pharmaceutical compositions of the present invention,any GFRA1-expressing cell can be damaged or killed. For example, breast,gastric, liver, renal or lung cancer cells are preferable as theGFRA1-expressing cells of the present invention. Of these, breastadenocarcinoma, breast carcinoma, adenosquamous carcinoma of thestomach, hepatocellular carcinoma (HCC), renal cell adenocarcinoma(RCC), or non-small cell lung cancer (NSCLC) cells are preferable.

Cells and antibodies can be contacted in vivo or in vitro. Whentargeting in vivo cancer cells as the GFRA1-expressing cells, themethods of the present invention are in fact therapeutic methods orpreventative methods for cancers. Specifically, the present inventionprovides therapeutic methods for cancers which comprise the followingsteps:

-   -   1) administering an antibody that binds GFRA1 to a cancer        patient, and    -   2) damaging cancer cells using the effector function of the        antibody bound to those cells.

The present inventors confirmed that antibodies binding GFRA1effectively damage GFRA1-expressing cells, in particular, breast,gastric, liver, renal or lung cancer cells using effector function. Thepresent inventors also confirmed that GFRA1 is highly expressed inbreast, gastric, liver, renal or lung cancer cells, with a highprobability. In addition, GFRA1 expression levels in normal tissues arelow. Putting this information together, methods of breast, gastric,liver, renal or lung cancer therapy where anti-GFRA1 antibody isadministered can be effective, with little danger of side effects.

The antibodies of the present invention are not limited so long as theycomprise a desired effector function. For example, antibodies comprisingthe Fc region of IgA, IgE, or IgG are essential for expressing ADCC.Equally, the antibody Fc region of IgM or IgG is preferable forexpressing CDC. Therefore, human-derived antibodies belonging to theseclasses are preferable in the present invention. Human antibodies can beacquired using antibody-producing cells harvested from humans, orchimeric animals transplanted with human antibody genes (Cloning andStem Cells., 4: 85-95, 2002).

Furthermore, antibody Fc regions can link with arbitrary variableregions. Specifically, chimeric antibodies wherein the variable regionsof different animal species are bound to human constant regions areknown. Alternatively, a human-human chimeric antibody can also beacquired by binding human-derived variable regions to arbitrary constantregions. In addition, CDR graft technology, where complementaritydetermining regions (CDRs) composing human antibody variable regions arereplaced with CDRs of heterologous antibodies, is also known(“Immunoglobulin genes”, Academic Press (London), pp 260-274, 1989;Proc. Natl. Acad. Sci. USA., 91: 969-973, 1994). By replacing CDRs,antibody binding specificity is replaced. That is, human GFRA1 will berecognized by humanized antibodies in which the CDR of humanGFRA1-binding antibodies has been transferred. The transferredantibodies can also be called humanized antibodies. Antibodiesthus-obtained and equipped with an Fc region essential to effectorfunction can be used as the antibodies of the present invention,regardless of the origin of their variable regions. For example,antibodies comprising a human IgG Fc are preferable in the presentinvention, even if their variable regions comprise an amino acidsequence derived from an immunoglobulin of another class or anotherspecies.

The antibodies of the present invention may be monoclonal antibodies orpolyclonal antibodies. Even when administering to humans, humanpolyclonal antibodies can be derived using the above-mentioned animalstransferred with a human antibody gene. Alternatively, immunoglobulinswhich have been constructed using genetic engineering techniques, suchas humanized antibodies, human-non-human chimeric antibodies, andhuman-human chimeric antibodies, can be used. Furthermore, methods forobtaining human monoclonal antibodies by cloning humanantibody-producing cells are also known.

GFRA1, or a fragment comprising its partial peptide, can be used asimmunogens to obtain the antibodies of the present invention. The GFRA1of the present invention can be derived from any species, preferablyfrom a mammal such as a human, mouse, or rat, and more preferably from ahuman. The human GFRA1 nucleotide sequence and amino acid sequence areknown (NM_(—)005264). The cDNA nucleotide sequence of GFRA1 is describedin SEQ ID NO: 1, and the amino acid sequences coded by that nucleotidesequence is described in SEQ ID NO: 2. One skilled in the art canroutinely isolate genes comprising the provided nucleotide sequence,preparing a fragment of the sequence as required, and obtain a proteincomprising the target amino acid sequence.

For example, the gene coding the GFRA1 protein or its fragment can beinserted into a known expression vector, and used to transform hostcells. The desired protein, or its fragment, can be collected frominside or outside host cells using arbitrary and standard methods, andcan also be used as an antigen. In addition, proteins, their lysates,and chemically-synthesized proteins can be used as antigens.Furthermore, cells expressing the GFRA1 protein or a fragment thereofcan themselves be used as immunogens.

When using a peptide fragment as the GFRA1 immunogen, it is particularlypreferable to select an amino acid sequence which comprises a regionpredicted to be an extra-cellular domain. The existence of a signalpeptide is predicted from positions 1 to 19 on the N-terminal of GFRA1(Jing S. et al., Cell. (1996) June 28; 85 (7):1113-24.). Thus, forexample, a region other than the N-terminal signal peptide (19 aminoacid residues) is preferred as the immunogen for obtaining theantibodies of the present invention. That is to say, antibodies thatbind to GFRA1 extra-cellular domains are preferred as the antibodies ofthe present invention.

Therefore, preferable antibodies in the present invention are antibodiesequipped with an Fc essential to effector function, and a variableregion that can bind to an extracellular GFRA1 domain. When aiming foradministration to humans, it is preferable to be equipped with an IgGFc.

Any mammal can be immunized with such an antigen. However, it ispreferable to consider compatibility with parent cells used in cellfusion. Generally, rodents, lagomorphs, or primates are used.

Rodents include, for example, mice, rats, and hamsters. Lagomorphsinclude, for example, rabbits. Primates include, for example, catarrhine(old world) monkeys such as Macaca fascicularis, Macaca mulatta, Sacredbaboons, and chimpanzees.

Methods for immunizing animals with antigens are well known in thefield. Intraperitoneal or subcutaneous antigen injections are standardmethods for immunizing mammals. Specifically, antigens can be dilutedand suspended in an appropriate amount of phosphate buffered saline(PBS), physiological saline, or so on. As desired, antigen suspensionscan be mixed with an appropriate amount of a standard adjuvant such asFreund's complete adjuvant, and administered to mammals afteremulsification. Subsequently, it is preferable that antigens mixed withan appropriate amount of Freund's incomplete adjuvant are administeredin multiple doses every four to 21 days. An appropriate carrier can alsobe used for immunization. After carrying out immunization as outlinedabove, standard methods can be used to examine serum for an increase inthe desired antibody level.

Polyclonal antibodies against the GFRA1 protein can be prepared fromimmunized mammals whose serum has been investigated for an increase inthe desired antibodies. This can be achieved by collecting blood fromthese animals, or by using an arbitrary, usual method to isolate serumfrom their blood. Polyclonal antibodies comprise serum that comprisespolyclonal antibodies, and fractions that comprise polyclonal antibodieswhich can be isolated from serum. IgG and IgM can be prepared fromfractions that recognize GFRA1 protein by using, for example, anaffinity column coupled to GFRA1 protein, and then further purifyingthis fraction using protein A or protein G columns. In the presentinvention, antiserum can be used as is as polyclonal antibodies.Alternatively, purified IgG, IgM, or such can also be used.

To prepare monoclonal antibodies, immunocytes are collected from mammalsimmunized with antigens, investigated for the increase of the desiredantibody level in serum (as above), and applied in cell fusion.Immunocytes for use in cell fusion preferably come from the spleen.Other preferred parent cells for fusion with the above immunogensinclude, for example, mammalian myeloma cells, and more preferably,myeloma cells that have acquired properties for selection of fusioncells by pharmaceutical agents.

The above immunocytes and myeloma cells can be fused using knownmethods, for example the methods of Milstein et al. (Galfre, G. andMilstein, C., Methods. Enzymol, 1981, 73, 3-46).

Hybridomas produced by cell fusion can be selected by culturing in astandard selective medium such as HAT medium (medium comprisinghypoxanthine, aminopterin, and thymidine). Cell culture in HAT medium isusually continued for several days to several weeks, a period sufficientenough to kill all cells other than the desired hybridomas (unfusedcells). Standard limiting dilutions are then carried out, and hybridomacells that produce the desired antibodies are screened and cloned.

Non-human animals can be immunized with antigens for preparinghybridomas in the above method. In addition, human lymphocytes fromcells infected with EB virus or such, can be immunized in vitro usingproteins, cells expressing proteins, or suspensions of the same. Theimmunized lymphocytes are then fused with human-derived myeloma cellsable to divide unlimitedly (U266 and so on), thus obtaining hybridomasthat produce the desired human antibodies which can bind the protein(Unexamined Published Japanese Patent Application No. (JP-A) Sho63-17688).

The obtained hybridomas are then transplanted to mice abdominalcavities, and ascites are extracted. The obtained monoclonal antibodiescan be purified using, for example, ammonium sulfate precipitation,protein A or protein G columns, DEAE ion exchange chromatography, oraffinity columns coupled to the proteins of the present invention. Theantibodies of the present invention can be used not only in purifyingand detecting the proteins of the present invention, but also ascandidates for agonists and antagonists of the proteins of the presentinvention. These antibodies can also be applied to antibody therapiesfor diseases related to the proteins of the present invention. When theobtained antibodies are administered to human bodies (antibody therapy),human antibodies or humanized antibodies are preferred due to their lowimmunogenicity.

For example, transgenic animals comprising a repertoire of humanantibody genes can be immunized with antigens selected from proteins,protein-expressing cells, or suspensions of the same. Antibody-producingcells are then recovered from the animals, fused with myeloma cells toyield hybridomas, and anti-protein human antibodies can be prepared fromthese hybridomas (see International Publication No. 92-03918, 93-2227,94-02602, 94-25585, 96-33735, and 96-34096).

Alternatively, immunocytes such as immunized lymphocytes that produceantibodies, can be immortalized using cancer genes, and used to preparemonoclonal antibodies.

Monoclonal antibodies obtained in this way can be prepared using methodsof genetic engineering (for example, see Borrebaeck, C. A. K. andLarrick, J. W., Therapeutic Monoclonal Antibodies, MacMillan Publishers,UK, 1990). For example, recombinant antibodies can be prepared bycloning DNAs that encode antibodies from immunocytes such as hybridomasor immunized lymphocytes that produce antibodies; then inserting theseDNAs into appropriate vectors; and transforming these into host cells.Recombinant antibodies prepared as above can also be used in the presentinvention.

The antibodies can be modified by binding with a variety of moleculessuch as polyethylene glycols (PEGs). Antibodies modified in this way canalso be used in the present invention. Modified antibodies can beobtained by chemically modifying antibodies. These kinds of modificationmethods are conventional to those skilled in the art. The antibodies canalso be modified by other proteins. Antibodies modified by proteinmolecules can be produced using genetic engineering. That is, targetproteins can be expressed by fusing antibody genes with genes that codefor modification proteins. For example, antibody effector function maybe enhanced on binding with cytokines or chemokines. In fact, theenhancement of antibody effector function for proteins fused with IL-2,GM-CSF, and such has been confirmed (Human Antibody, 10: 43-49, 2000).IL-2, IL-12, GM-CSF, TNF, eosinophil chemotactic substance (RANTES) andso on can be included in cytokines or chemokines that enhance effectorfunction.

Alternatively, antibodies of the present invention can be obtained aschimeric antibodies which comprise a non-human antibody-derived variableregion and a human antibody-derived constant region, or as humanizedantibodies which comprise a non-human antibody-derived complementaritydetermining region (CDR), a human antibody-derived framework region(FR), and a constant region. Such antibodies can be produced using knownmethods.

Antibodies obtained as above can be purified until uniform. For example,antibodies can be purified or separated according to general methodsused for purifying and separating proteins. For example, antibodies canbe separated and isolated using appropriately selected combinations ofcolumn chromatography, comprising but not limited to affinitychromatography, filtration, ultrafiltration, salt precipitation,dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing,and so on (Antibodies: A Laboratory Manual, Harlow and David, Lane(edit.), Cold Spring Harbor Laboratory, 1988).

Protein A columns and Protein G columns can be used as affinity columns.Exemplary protein A columns in use include Hyper D, POROS, and SepharoseF.F (Pharmacia).

Exemplary chromatography (excluding affinity chromatography) include ionexchange chromatography, hydrophobic chromatography, gel filtration,reverse phase chromatography, and adsorption chromatography (“Strategiesfor Protein Purification and Characterization: A Laboratory CourseManual” Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press,1996). The chromatography can be performed according to the procedure ofliquid phase chromatographies such as HPLC or FPLC.

For example, the antigen-binding activity of the antibodies of thepresent invention can be measured by using absorbance measurements,enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA),radioimmunoassays (RIA) and/or immunofluorescence methods. In ELISA, anantibody of the present invention is immobilized on a plate, a proteinof the present invention is added to the plate, and then a samplecomprising the desired antibody such as the culture supernatant of cellsthat produce the antibody or purified antibody is added. A secondaryantibody that recognizes the primary antibody and has been tagged withan enzyme such as alkaline phosphatase is then added, and the plate isincubated. After washing, an enzyme substrate such as p-nitrophenylphosphate is added to the plate, absorbance is measured, and theantigen-binding activity of the samples is evaluated. Protein fragments(C-terminal or N-terminal fragments, and such) can be used in the sameway as proteins. The binding activity of the antibodies of the presentinvention can be evaluated using BIAcore (Pharmacia).

In addition, by following the methods outlined in the Examples, antibodyeffector function can also be evaluated. For example, targetGFRA1-expressing cells are incubated with effector cells in the presenceof an antibody whose effector function is to be evaluated. If targetcell destruction is detected, the antibody can be confirmed to compriseeffector function that induces ADCC. The level of observed target celldestruction, in the absence of either antibodies or effector cells, canbe compared as a control with the level of effector function. Cellswhich clearly express GFRA1 can be used as the target cells.Specifically, a variety of cell lines confirmed to express GFRA1 in theExamples can be used. These cell lines can be obtained from cell banks.In addition, monoclonal antibodies which comprise more powerful effectorfunction can be selected.

In the present invention, anti-GFRA1 antibodies can be administered tohumans or other animals as pharmaceutical agents. In the presentinvention, animals other than humans to which the antibodies can beadministered include mice, rats, guinea pigs, rabbits, chickens, cats,dogs, sheep, pigs, cows, monkeys, baboons, and chimpanzees. Theantibodies can be directly administered to subjects, and in addition,can be formulated into dosage forms using known pharmaceuticalformulation methods. For example, depending on requirements, they can beparenterally administered in an injectable form such as a sterilesolution or suspension with water or other arbitrary pharmaceuticallyacceptable fluid. For example, this kind of compounds can be mixed withacceptable carriers or solvents, specifically sterile water,physiological saline, vegetable oils, emulsifiers, suspension agents,surfactants, stabilizers, flavoring agents, excipients, solvents,preservatives, binding agents and the like, into a generally acceptedunit dosage essential for use as a pharmaceutical agent.

Other isotonic solutions comprising physiological saline, glucose, andadjuvants (such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride) can be used as the injectable aqueous solution. They can alsobe used with appropriate solubilizers such as alcohols, specificallyethanols and polyalcohols (for example, propylene glycols andpolyethylene glycol), and non-ionic surfactants (for example polysorbate80™ or HCO-50).

Sesame oils or soybean oils can be used as an oleaginous solution, andbenzyl benzoate or benzyl alcohols can be used with them as asolubilizer. Buffer solutions (phosphate buffers, sodium acetatebuffers, or so on), analgesics (procaine hydrochloride or such),stabilizers (benzyl alcohol, phenols, or so on), and antioxidants can beused in the formulation. The prepared injections can be packaged intoappropriate ampules.

In the present invention, the anti-GFRA1 antibodies can be administeredto patients, for example, intraarterially, intravenously, orpercutaneously, or intranasally, transbronchially, locally, orintramuscularly. Intravascular (intravenous) administration by drip orinjection is an example of a general method for systematicadministration of antibodies to breast, gastric, liver, renal or lungcancer patients. Methods of locally concentrating antibody agents to theprimary focus or metastatic focus in the lung include local injectionusing a bronchoscope (bronchoscopy) and local injection under CTguidance or with thoracoscopy. Methods of locally concentrating antibodyagents to the primary focus or metastatic focus in the liver includelocal injection using a hepatic portal injection or arterial infusion.In addition, methods in which an intraarterial catheter is inserted neara vein that supplies nutrients to cancer cells to locally injectanti-cancer agents such as antibody agents, are effective as localcontrol therapies for metastatic focuses as well as primary focuses ofbreast, gastric, liver, renal or lung cancer.

Although dosage and administration methods vary according to patientbody weight and age, and administration method, these can be routinelyselected by one skilled in the art. In addition, DNA encoding anantibody can be inserted into a vector for gene therapy, and the vectorcan be administered for therapy. Dosage and administration methods varyaccording to patient body weight, age, and condition, however, oneskilled in the art can select these appropriately.

Anti-GFRA1 antibodies can be administered to living bodies in an amountsuch that cytotoxicity based on effector function againstGFRA1-expressing cells can be confirmed. For example, although there isa certain amount of difference depending on symptoms, anti-GFRA1antibody dosage is 0.1 mg to 250 mg/kg per day. Usually, the dosage foran adult (of weight 60 kg) is 5 mg to 17.5 g/day, preferably 5 mg to 10g/day, and more preferably 100 mg to 3 g/day. The dosage schedule isfrom one to ten times over a two to ten day interval, and for example,progress is observed after a three to six times administration.

Although the antibodies of the invention retain effector function, insome embodiments, cytotoxic agents can be linked to the antibodies usingwell known techniques. Cytotoxic agents are numerous and varied andinclude, but are not limited to, cytotoxic drugs or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diphtheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin, auristatin and thelike. Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to the antibodies of the invention or binding of aradionuclide to a chelating agent that has been covalently attached tothe antibody. Methods for preparing such conjugates are well known inthe art.

In addition, the present invention provides immunogenic compositions forinducing antibodies comprising effector functions againstGFRA1-expressing cells, where the compositions comprise as an activeingredient GFRA1 or an immunologically active GFRA1 fragment, or a DNAor cell which can express the same. Alternatively, the present inventionrelates to uses of GFRA1 or an immunologically active GFRA1 fragment, ora DNA or cell which can express the same in the production ofimmunogenic compositions for inducing antibodies comprising effectorfunctions against GFRA1-expressing cells.

The administration of anti-GFRA1 antibodies damages cancer cells by theeffector function of those antibodies. Thus, if GFRA1 antibodies can beinduced in vivo, therapeutic effects equivalent to the antibodyadministration can be achieved. When administering immunogeniccompositions comprising antigens, target antibodies can be induced invivo. The immunogenic compositions of the present invention thus areparticularly useful in vaccine therapy against GFRA1-expressing cells.Thus, the immunogenic compositions of the present invention areeffective as, for example, vaccine compositions for breast, gastric,liver, renal or lung cancer therapies.

The immunogenic compositions of the present invention can comprise GFRA1or an immunologically active GFRA1 fragment, as an active ingredient. Animmunologically active GFRA1 fragment refers to a fragment that caninduce anti-GFRA1 antibodies which recognize GFRA1 and comprise effectorfunction. Below, GFRA1 and the immunologically active GFRA1 fragment aredescribed as immunogenic proteins. Whether a given fragment inducestarget antibodies can be determined by actually immunizing an animal,and confirming the activity of the induced antibodies. Antibodyinduction and the confirmation of its activity can be carried out, forexample, using methods described in Examples. For example, fragmentscomprising an amino acid sequence corresponding to GFRA1 position 24 to465 can be used as the immunogens of the present invention.

The immunogenic compositions of the present invention comprisepharmaceutically acceptable carriers as well as immunogenic proteins,the active ingredients. If necessary, the compositions can also becombined with an adjuvant. Killed tuberculosis bacteria, diphtheriatoxoid, saponin and so on can be used as the adjuvant.

Alternatively, DNAs coding for the immunogenic proteins, or cellsretaining those DNAs in an expressible state, can be used as theimmunogenic compositions. Methods for using DNAs expressing the targetantigen as immunogens, so-called DNA vaccines, are well known. DNAvaccines can be obtained by inserting a DNA encoding GFRA1 or itsfragment into an appropriate expression vector.

Retrovirus vectors, adenovirus vectors, adeno-associated virus vectors,Sendai virus vectors or such can be used as the vector. In addition,DNAs in which a DNA encoding an immunogenic protein is functionallyconnected downstream of a promoter can be directly introduced into cellsas naked DNA, and then expressed. Naked DNA can be encapsulated inribosomes or viral envelope vectors and introduced into cells.

The GFRA1 polypeptides and polynucleotides of the invention can also beused for the induction of an immune response in vivo, includingproduction of antibodies and cytotoxic T lymphocytes (CTL) specific forGFRA1 expressing cells. In such methods, CTL induction by a desiredpeptide can be achieved by presenting the peptide to a T cell via anantigen presenting cell (APC) either in vivo or ex vivo.

For example, patient blood cells e.g., peripheral blood mononuclearcells (PBMC) are collected, transformed using a vector that can expressthe immunogenic proteins, and returned to the patient. Transformedbloods cells produce the immunogenic proteins inside the body of thepatient, and induce the target antibodies. Alternatively, PBMCs of thepatient are collected, the cells are contacted with the polypeptide exvivo, and following the induction of APCs or CTLs, the cells may beadministered to the subject. APCs or CTLs induced in vitro can be clonedprior to administration. By cloning and growing cells having highactivity of damaging target cells, cellular immunotherapy can beperformed more effectively. Furthermore, APCs and CTLs isolated in thismanner may be used for cellular immunotherapy not only againstindividuals from whom the cells are derived, but also against similartypes of tumors from other individuals.

Generally, when using a polypeptide for cellular immunotherapy,efficiency of the CTL-induction is known to be increased by combining aplurality of polypeptides having different structures and contactingthem with APCs, particularly, dendritic cells. Therefore, whenstimulating APCs with protein fragments, it is advantageous to use amixture of multiple types of fragments.

The induction of anti-tumor immunity by a polypeptide can be confirmedby observing the induction of antibody production against tumors. Forexample, when antibodies against a polypeptide are induced in alaboratory animal immunized with the polypeptide, and when growth oftumor cells is suppressed by those antibodies, the polypeptide is deemedto have the ability to induce anti-tumor immunity.

When DNAs encoding the immunogenic proteins, or cells transformed withthe same are used as immunogenic compositions of the present invention,they can be combined with immunogenic proteins as well as carrierproteins that enhance their immunogenic properties.

As noted above, the present invention provides methods for inducingantibodies which comprise effector function against GFRA1-expressingcells, where the methods comprise the step of administering GFRA1, animmunologically active GFRA1 fragment, or DNA or cells that can expressthe same. The methods of the present invention induce antibodies thatcomprise effector function that damages GFRA1-expressing cells such asbreast, gastric, liver, renal or lung cancers. As a result, therapeuticeffects for breast, gastric, liver, renal or lung cancers and so on canbe obtained.

Each day, 0.1 mg to 250 mg per kilogram of the immunogenic compositionsof the present invention can be administered orally or parenterally.Parenteral administration includes subcutaneous injection andintravenous injection. The administrative dose for a single adult isusually 5 mg to 17.5 g/day, preferably 5 mg to 10 g/day, and morepreferably 100 mg to 3 g/day.

All prior art references cited herein are incorporated by reference intheir entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are photographs depicting the result of Semiquantitative RT-PCRanalysis for the GFRA1 gene in cancer cells. A; for breast cancer celllines. B; for gastric cancer cell lines. C, for liver cancer cell lines.D, for renal cancer cell lines. E, for lung cancer cell lines. Theexpression level of harceptin target gene c-erbB2 gene for breast canceris indicated in panel A (positive control).

FIG. 2 shows the results of an ADCC assay using Herceptin against (A)MDA-MB-453 over-expressed c-erbB-2 gene and (B) MCF-7 low-expressedc-erbB-2 gene.

FIG. 3 shows the results of an ADCC assay using anti-GFRA1 antibodyBr003 against GFRA1-over- and low-expressing breast cancer cell line,(A) MCF-7 and (B) MDA-MB-453, respectively.

FIG. 4 shows the results of an ADCC assay using anti-GFRA1 antibodyBr003 against GFRA1-over-expressing gastric cancer cell line, MKN1.

FIG. 5 shows the results of an ADCC assay using anti-GFRA1 antibodyBr003 against GFRA1-over-expressing liver cancer cell line, SNU-398.

FIG. 6 shows the results of an ADCC assay using anti-GFRA1 antibodyBr003 against GFRA1-over-expressing renal cancer cell line, ACHN.

FIG. 7 shows the results of an ADCC assay using anti-GFRA1 antibodyBr003 against GFRA1-over-expressing lung cancer cell line, NCI-H1793.

BEST MODE FOR CARRYING OUT THE INVENTION

Below, the present invention is further explained based on Examples.

Cell Line:

Human breast, gastric, liver, renal or lung cancer cell lines werepropagated as a monolayer in an appropriate medium with 10% fetal bovineserum. The cell lines used in the experiment are shown in Table 1.

TABLE 1 Cell line Medium Place obtained Breast cancer Cell line BT-20E-MEM*¹ + 10% FBS American Type Culture Collection (ATCC); HTB-19 BT-474D-MEM*² + 10% FBS ATCC; HTB-20 BT-549 RPMI + 10% FBS ATCC; HTB-122HCC1143 RPMI + 10% FBS ATCC; CRL-2321 HCC1395 RPMI + 10% FBS + 2 mMATCC; CRL-2324 L-glutamin HCC1500 RPMI + 10% FBS + 2 mM ATCC; CRL-2329L-glutamin HCC1937 RPMI + 10% FBS + 2 mM ATCC; CRL-2336 L-glutamin MCF-7E-MEM*¹ + 10% FBS ATCC; HTB-22 MDA-MB-157 L15*³ + 10% FBS ATCC; HTB-24MDA-MB-231 L15*³ + 10% FBS ATCC; HTB-26 MDA-MB-435S L15*³ + 10% FBSATCC; HTB-129 MDA-MB-453 McCoy*⁴ + 10% FBS ATCC; HTB-131 SK-BR-3 RPMI +10% FBS ATCC; HTB-30 T-47D RPMI + 10% FBS + 2mM ATCC; HTB-133 L-glutaminZR-75-1 E-MEM*¹ + 10% FBS ATCC; CRL-1500 Gastric cancer Cell line MKN1RPMI + 10% FBS Health Science Research Resources Bank (HSRRB); JCRB0252MKN7 RPMI + 10% FBS HSRRB; JCRB1025 MKN45 RPMI + 10% FBS HSRRB; JCRB0254MKN74 RPMI + 10% FBS HSRRB; JCRB0255 Liver cancer Cell line AlexanderD-MEM*² + 10% FBS HSRRB; IFO50069 Hep G2 D-MEM*² + 10% FBS HSRRB;JCRB1054 HUH-6 Clone 5 E-MEM*¹ + 10% FBS HSRRB; JCRB0401 HuH-7 D-MEM*² +10% FBS HSRRB; JCRB0403 SNU-398 RPMI + 10% FBS ATCC; CRL-2233 (heatinactivated) SNU-423 RPMI + 10% FBS ATCC; CRL-2238 SNU-449 RPMI + 10%FBS ATCC; CRL-2234 SNU-475 RPMI + 10% FBS ATCC; CRL-2236 Renal cancerCell line ACHN RPMI + 5% FBS ATCC; CRL-1611 786-O RPMI + 10% FBS ATCC;CRL-1932 A-498 E-MEM*¹ + 10% FBS ATCC; HTB-44 Caki-1 McCoy*⁴ + 10% FBSHSRRB; JCRB0801 Caki-2 McCoy*⁴ + 10% FBS ATCC; HTB-47 Lung cancer Cellline NCI-H23 RPMI + 10% FBS ATCC; CRL-5800 NCI-H358 RPMI + 10% FBS ATCC;CRL-5807 NCI-H596 RPMI + 10% FBS ATCC; HTB-178 NCI-H1650 RPMI + 10% FBSATCC; CRL-5883 NCI-H1793 F12*⁵ + D-MEM*² + 10% FBS ATCC; CRL-5896 PC-14RPMI + 10% FBS RIKEN Bioresource Center SK-MES-1 E-MEM*¹ + 10% FBS + 2mM ATCC; HTB-58 L-glutamin SK-LU-1 E-MEM*¹ + 10% FBS + 2 mM ATCC; HTB-57L-glutamin SW900 L15*³ + 10% FBS ATCC; HTB-59 SW1573 L15*³ + 10% FBSATCC; CRL-2170 A549 RPMI + 10% FBS ATCC; CCL-185 NCI-H522 RPMI + 10% FBSATCC; CRL-5810 PC-3 E-MEM*¹ + 10% FBS HSRRB; JCRB0077 *¹Eagle's MinimalEssential medium *²Dulbecco's Modified Eagle's medium *³Leibovitz's L-15medium *⁴McCoy's 5A medium Modified *⁵F-12 Nutrient Mixture (HAM)

Furthermore, the following cell lines were used in ADCC assays usinganti-GFRA1 antibodies:

Breast adenocarcinoma (BC): MCF-7Breast carcinoma: MDA-MB-453.Stomach adenosquamous carcinoma: MKN1.Hepatocellular carcinoma (HCC): SNU-398.Renal cell adenocarcinoma (RCC): ACHN.Non-small cell lung carcinoma (NSCLC): NCI-H1793.

Antibody Production:

According to standard protocols, individual protein specific antibodieswere produced using His-tagged fusion proteins expressed in bacteria asimmunogens. These fusion proteins comprised a protein portion thatcorresponded to one part of the protein (residues 24 to 465).

Semiquantitative RT-PCR for GFRA1 and c-erbB2:

Total RNA was extracted from the cell lines using the Rneasy® Kit(QIAGEN). In addition, mRNA was purified from total RNA by Oligo(dT)-cellulose column (Amersham Biosciences) and synthesized tofirst-strand cDNA by reverse transcription (RT) using the SuperScriptFirst-Strand Synthesis System (Invitrogen). It was prepared appropriatedilutions of each first-stranded cDNA for subsequent PCR amplificationby monitoring GAPDH as a quantitative control. The primer sequences thepresent inventors used were 5′-CTGAAGCAGAAGTCGCTCTA-3′ (SEQ.ID.NO.3) and5′-GACAGCTGCTGACAGACCTT-3′ (SEQ.ID.NO.4) for GFRA1,5′-GTCAGTGGTGGACCTGACCT-3′ (SEQ.ID.NO.5) and5′-GGTTGAGCACAGGGTACTTTATT-3′ (SEQ.ID.NO.6) for GAPDH. All PCR reactionsinvolved initial denaturation at 94° C. for 2 min and consisted of 94°C. for 30 s, 58° C. for 30 s, and 72° C. for 1 min by 21 cycles (forGAPDH) or 30-40 cycles (for GFRA1), on a GeneAmp PCR system 9700 (PEApplied Biosystems).

The over-expression of GFRA1 was found in breast cancer cell line MCF-7(FIG. 1). In addition, to elucidate the efficacy of anti-GFRA1 antibody(Br003) on various cancers, the expression of GFRA1 was confirmed. Theover-expression of GFRA1 was decided in gastric cancer cell line MKN1,liver cancer cell line SNU-398, renal cancer cell line ACHN, and lungcancer cell line NCI-H1793.

Flow Cytometry Analysis:

Cancer cells (5×10⁶) were incubated at 4° C. for 30 minutes with thepurified polyclonal antibodies (pAb) or rabbit IgG (the control). Cellswere washed with phosphate buffer solution (PBS) and then incubated at4° C. for 30 minutes in FITC-labeled Alexa Fluor 488. The cells wereagain washed in PBS, and analyzed on a flow cytometer (FACSCalibur®,Becton Dickinson) and then analyzed by BD CellQuest™ Pro software(Becton Dickinson). Mean fluorescence intensity (MFI) was defined as aratio of the flow cytometric intensity (intensity by each proteinspecific antibody/intensity by rabbit IgG).

Using anti-GFRA1 antibodies Br003, GFRA1 expression was investigated forMCF-7, MKN1, SNU-398, ACHN, and NCI-H1793 cells. As a result, a higherproportion of anti-GFRA1 antibodies (BrO03) bound to MCF-7, MKN1,SNU-398, ACHN, and NCI-H1793 cells (MFI (Mean fluorescence intensity):155.4, 5.2, 9.3, 78.5, and 9.4, respectively) than did rabbit IgG (thecontrol).

ADCC Assays:

Target cells were exposed with 0.8 μM of calcein acetoxymethyl estel(Calcein-AM, DOJINDO) at 37° C. for 30 minutes. Calcein-AM becomesfluorescent after the cleavage of calcein-AM by cellular esterases thatproduce a fluorescent derivate calcein. Target cancer cells were washedtwo times before being added to the assay, and cells were then plated on96-well U-bottom plates (4×10³ cells/well). Human peripheral bloodmononuclear cells (PMBC) were harvested from a healthy person, separatedusing Ficoll-Paque (Amersham Biosciences) density gradientcentrifugation, and then used as effector cells. Target cancer cells (T)and effector cells (E) were co-incubated in 200 μl of AIM-V medium in96-well U-bottom plates at various E:T ratios (50:1, 25:1, 12.5:1, and6.25:1) with Br003 anti-GFRA1 antibody (2 □g/well) or control antibodyHerceptin (2 □g/well, Roche). This incubation was carried out intriplicate, in 200 μL of AIM-V medium (Life Technologies, Inc), at 37°C. for six hours. Control assays included the incubation of target cellswith only anti-GFRA1 antibody Br003 or effector cells. Herceptin wasused as a control in some experiments.

The ADCC effects of anti-GFRA1 antibody (Br003) for MCF-7, MKN1,SNU-398, ACHN, and NCI-H1793 cells were evaluated based on thefluorescent images of viable cells were rapidly acquired using the INCell Analyzer 1000 (Amersham Bioscience). These images were numericallyconverted as viable cell count by counting the fluorescent object orvesicle using Developer tool ver. 5.21 software (Amersham Bioscience).Control assays were carried out by incubating target cells with onlyBr003 anti-GFRA1 antibody or only effector cells. Herceptin was used asa control in several experiments (FIG. 2A,2B). Direct cell damage ofMCF-7, MKN1, SNU-398, ACHN, and NCI-H1793 cells by Br003 anti-GFRA1antibody itself was not observed. However, Br003 induced ADCC in MCF-7,MKN1, SNU-398, ACHN, and NCI-H1793 cells that over-expressed GFRA1 (FIG.3A,4-7), while no effect against MDA-MB-453 cells with GFRA1low-expression (FIG. 3B).

INDUSTRIAL APPLICABILITY

The present invention is based, at least in part, on the discovery thatGFRA1-expressing cells can be damaged by antibody cytotoxicity. GFRA1was identified by the present inventors as a gene strongly expressed inbreast, gastric, liver, renal or lung cancers. Thus, treatment ofdisease associated with GFRA1-expressing cells, for example, breast,gastric, liver, renal or lung cancer is conveniently carried out usingantibodies that bind to GFRA1. Results actually confirmed by the presentinventors show cytotoxicity due to the effect of ADCC in breast,gastric, liver, renal or lung cancer cell lines, in the presence ofGFRA1 antibodies.

1. A pharmaceutical composition for damaging a GFRA1-expressing cell,the composition comprising an anti-GFRA1 antibody as an activeingredient, wherein the antibody comprises antibody effector function.2. The pharmaceutical composition of claim 1, wherein theGFRA1-expressing cell is a breast, gastric, liver, renal or lung cancercell.
 3. The pharmaceutical composition of claim 1, wherein theanti-GFRA1 antibody is a monoclonal antibody.
 4. The pharmaceuticalcomposition of claim 1, wherein the antibody effector function is eitherantibody-dependent cytotoxicity or complement-dependent cytotoxicity, orboth.
 5. A method for damaging a GFRA1-expressing cell, comprising thesteps of: a) contacting the GFRA1-expressing cell with an anti-GFRA1antibody, and b) damaging the GFRA1-expressing cell with the effectorfunction of the antibody that has bound to the cell.
 6. An immunogeniccomposition for inducing an antibody that comprises an effector functionagainst a GFRA1-expressing cell, wherein the composition comprises, asan active ingredient, GFRA1, an immunologically active fragment thereof,or a DNA that can express GFRA1 or the immunoligically active fragment.7. A method for inducing an antibody that comprises an effector functionagainst an GFRA1-expressing cell, wherein the method comprisesadministering GFRA1, an immunologically active fragment thereof, or acell or a DNA that can express GFRA1 or the immunoligically activefragment.